Method of synthesizing peptides, proteins and bioconjugates

Abstract

The invention relates to the synthesis of peptides, proteins and related bioconjugates, and in particular, to such synthesis using a peptide ligation method whereby a C-terminal salicylaldehyde ester peptide is reacted with an aminoacyl-N-hydroxl peptide. The invention also relates to the synthesis of cyclic peptides, including serinyl- or threonyl-containing cyclic peptides. The invention further relates to a solid phase synthesis of C-terminal salicylaldehyde ester peptides.

The invention claimed is: 
     
       1. A method of preparing a polypeptide or protein of formula (3) 
       
         
           
           
               
               
           
         
       
       comprising:
 reacting a C-terminal salicylaldehyde ester peptide of formula (1) 
 
       
         
           
           
               
               
           
         
         with an aminoacyl-N-hydroxyl peptide of formula (2) 
       
       
         
           
           
               
               
           
         
         wherein the reaction is carried out in a pyridine/organic acid mixture to form an intermediate compound having a 1,2,5-oxadiazinane ring structure; and 
         adding a reducing agent to cleave the 1,2,5-oxadiazinane ring structure of the intermediate compound to form the polypeptide or protein of formula (3), wherein each of R 1 , R 2  and R 3  is independently selected from the group consisting of H, a substituted or unsubstituted C 1 -C 10  alkyl, substituted or unsubstituted C 2 -C 15  alkenyl, substituted or unsubstituted C 2 -C 15  alkynyl, substituted or unsubstituted C 3 -C 15  cycloalkyl, substituted or unsubstituted C 3 -C 15  cycloalkenyl, substituted or unsubstituted C 3 -C 15  heterocycloalkyl, substituted or unsubstituted C 3 -C 15  heterocycloalkenyl, substituted or unsubstituted C 6 -C 15  aryl, or substituted or unsubstituted C 6 -C 15  heteroaryl, and a side chain of an amino acid; or 
         R 1  is C 1 -C 10  alkylyl that forms together with the nitrogen of the adjacent backbone imino group, a heteroalicyclic ring. 
       
     
     
       2. The method of  claim 1 , wherein at least one of R 1  and R 3  is independently selected from the group consisting of H, CH 3 , CH(CH 3 ) 2 , CH 2 CH(CH 3 ) 2 , CH(CH 3 )CH 2 CH 3 , (CH 2 ) 2 —S—CH 3 , CH 2 —SH, CH 2 —OH, CHOH—CH 3 , CH 2 -(3-indolyl), CH 2 -phenyl, (CH 2 ) 4 —NH 2 , (CH 2 ) 3 —NH—C(—NH)NH 2 , CH 2 -1H-imidazol-4-yl, CH 2 -(p-hydroxy-phenyl), CH 2 —C(O)NH 2 , (CH 2 ) 2 —C(O)NH 2 , CH 2 —COOH, and (CH 2 ) 2 —COOH, or R 1  is (CH 2 ) 3  that forms together with the nitrogen of the adjacent backbone imino group a 5-membered heteroalicyclic ring. 
     
     
       3. The method of  claim 1 , wherein R 2  is selected from the group consisting of H, CH 3 , CH(CH 3 ) 2 , CH 2 CH(CH 3 ) 2 , CH(CH 3 )CH 2 CH 3 , (CH 2 ) 2 —S—CH 3 , CH 2 —SH, CH 2 —OH, CHOH—CH 3 , CH 2 -(3-indolyl), CH 2 -phenyl, (CH 2 ) 4 —NH 2 , (CH 2 ) 3 —NH—C(═NH)NH 2 , CH 2 -1H-imidazol-4-yl, CH 2 -(p-hydroxy-phenyl), CH 2 —C(O)NH 2 , (CH 2 ) 2 —C(O)NH 2 , CH 2 —COOH, and (CH 2 ) 2 —COOH. 
     
     
       4. The method of  claim 1 , wherein the reducing agent is samarium (II) iodide (SmI 2 ) solution. 
     
     
       5. The method of  claim 4 , wherein the reducing agent is SmI 2  solution in tetrahydrofuran (THF) or SmI 2  solution in trifluoroethanol (TFE). 
     
     
       6. The method of  claim 1 , wherein the organic acid is acetic acid, propionic acid, isobutyric acid or butyric acid. 
     
     
       7. The method of  claim 1 , wherein the intermediate compound has the following formula (4): 
       
         
           
           
               
               
           
         
       
     
     
       8. The method of  claim 2 , wherein at least one of R 1 , R 2  and R 3  is independently selected from the group consisting of CH 3 , CH 2 -phenyl, CH 2 CH(CH 3 ) 2 , CH(CH 3 ) 2  and H. 
     
     
       9. The method of  claim 2 , wherein:
 R 1  is CH 3  and R 2  is CH 3 ; 
 R 1  is CH 3  and R 2  is H; 
 R 1  is CH 3  and R 2  is CH 2 -phenyl; 
 R 1  is CH 3  and R 2  is CH 2 CH(CH 3 ) 2 ; 
 R 1  is CH 2 -phenyl and R 2  is CH 3 ; 
 R 1  is CH 2 CH(CH 3 ) 2  and R 2  is CH 3 ; 
 R 1  is CH(CH 3 ) 2  and R 2  is CH 3 ; 
 R 1  is CH(CH 3 ) 2  and R 2  is H; 
 R 1  is CH 2 CH(CH 3 ) 2  and R 2  is CH 2 CH(CH 3 ) 2 ; or 
 R 1  is CH 2 -phenyl and R 2  is CH 2 CH(CH 3 ) 2 . 
 
     
     
       10. A method of preparing a cyclic peptide of formula (5) 
       
         
           
           
               
               
           
         
         where AA n  represents an amino acid at a n th  position of the peptide, comprising: 
         reacting the C-terminal salicylaldehyde ester peptide of formula (1A) 
       
       
         
           
           
               
               
           
         
       
       in a pyridine/organic acid mixture, where “ - - - ” represents additional amino acids in the chain of the peptide; and
 adding a reducing agent to form the cyclic peptide of formula (5). 
 
     
     
       11. The method of  claim 10 , wherein the reducing agent is SmI 2  solution. 
     
     
       12. The method of  claim 10 , wherein the reducing agent is SmI 2  solution in THF or SmI 2  solution in TFE. 
     
     
       13. The method of  claim 10 , wherein the organic acid is acetic acid, propionic acid, isobutyric acid or butyric acid. 
     
     
       14. A method of preparing a serinyl- or threonyl-containing cyclic peptide of formula (6) 
       
         
           
           
               
               
           
         
         where the peptide contains serine (Ser) or threonine (Thr), comprising: 
         reacting the C-terminal Ser- or Thr-containing salicylaldehyde ester peptide of formula (1B) 
       
       
         
           
           
               
               
           
         
         in a pyridine/organic acid mixture; and 
         adding an acidic deprotecting agent to form the serinyl- or threonyl-containing cyclic peptide of formula (6), 
         wherein R is a substituted or unsubstituted C 1 -C 10  alkyl, substituted or unsubstituted C 2 -C 15  alkenyl, substituted or unsubstituted C 2 -C 15  alkynyl, substituted or unsubstituted C 3 -C 15  cycloalkyl, substituted or unsubstituted C 3 -C 15  cycloalkenyl, substituted or unsubstituted C 3 -C 15  heterocycloalkyl, substituted or unsubstituted C 3 -C 15  heterocycloalkenyl, substituted or unsubstituted C 6 -C 15  aryl, or substituted or unsubstituted C 6 -C 15  heteroaryl, or a side chain of an amino acid. 
       
     
     
       15. The method of  claim 14 , wherein the acidic deprotecting agent to deprotect the oxazolidine ring is trifluoroacetic acid (TFA). 
     
     
       16. The method of  claim 14 , wherein the organic acid is acetic acid, propionic acid, isobutyric acid, or butyric acid. 
     
     
       17. A method of preparing a C-terminal salicylaldehyde ester peptide in solid phase, comprising:
 (i) mixing 2-hydroxycinnamic acid and 4-methylbenzhydrylamine hydrochloride salt (MBHA) resin in the presence of benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate (PyBOP) and a tertiary amine to form a 2-hydroxycinnamamide linker on the resin; 
 (ii) coupling a Boc-amino acid to the 2-hydroxycinnamamide linker-resin, followed by standard Boc solid phase peptide synthesis to form a peptidyl 2-hydroxycinnamamide phenolic ester on the resin, where Boc represents tert-butoxycarbonyl protecting group; 
 (iii) adding a cleavage agent to release the peptidyl 2-hydroxycinnamamide phenolic ester from the resin; and 
 (iv) ozonizing the C═C bond of the peptidyl 2-hydroxycinnamamide phenolic ester to obtain the C-terminal salicylaldehyde ester peptide. 
 
     
     
       18. The method of  claim 17 , wherein the tertiary amine is diisopropylethylamine (DIEA). 
     
     
       19. The method of  claim 17 , wherein the cleavage agent is hydrofluoric acid or trifluoromethanesulfonic acid-trifluoroacetic acid mixture. 
     
     
       20. A method of preparing a polypeptide or protein of formula (7) 
       
         
           
           
               
               
           
         
         comprising reacting a compound of formula (4) 
       
       
         
           
           
               
               
           
         
         with trifluoroacetic acid (TFA), 
         wherein each of R 1 , R 2  and R 3  is independently selected from the group consisting of H, a substituted or unsubstituted C 1 -C 10  alkyl, substituted or unsubstituted C 2 -C 15  alkenyl, substituted or unsubstituted C 2 -C 15  alkynyl, substituted or unsubstituted C 3 -C 15  cycloalkyl, substituted or unsubstituted C 3 -C 15  cycloalkenyl, substituted or unsubstituted C 3 -C 15  heterocycloalkyl, substituted or unsubstituted C 3 -C 15  heterocycloalkenyl, substituted or unsubstituted C 6 -C 15  aryl, or substituted or unsubstituted C 6 -C 15  heteroaryl, and a side chain of an amino acid.